Bioclimatic analysis and spatial distribution of fascioliasis causative agents by assessment of Lymnaeidae snails in northwestern provinces of Iran

Background Snails of the Lymnaeidae family are the intermediate hosts of Fasciola species, the causative agents of fascioliasis. The purpose of this study was to determine the prevalence of Fasciola species in lymnaeid snails and to investigate the association of geoclimatic factors and Fasciola species distribution in northwestern provinces of Iran using geographical information system (GIS) data. Methods A total of 2000 lymnaeid snails were collected from 33 permanent and seasonal habitats in northwestern Iran during the period from June to November 2021. After identification by standard morphological keys, they were subjected to shedding and crushing methods. Different stages of Fasciola obtained from these snails were subjected to the ITS1 polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method for species identification. The associations of weather temperature, rainfall, humidity, evaporation, air pressure, wind speed, elevation, and land cover with the distribution of Fasciola species were investigated. Geographical and statistical analysis was performed using ArcMap and SPSS software, respectively, to determine factors related to Fasciola species distribution. Results Of the 2000 snails collected, 19 were infected with Fasciola hepatica (0.09%), six with F. gigantica (0.03%), and 13 with other trematodes. Among geoclimatic and environmental factors, mean humidity, maximum humidity, and wind speed were significantly higher in areas where F. hepatica was more common than F. gigantica. The altitude of F. hepatica-prevalent areas was generally lower than F. gigantica areas. No significant relationship was observed between other investigated geoclimatic factors and the distribution of infected snails. Conclusions The present study showed the relationship of humidity and wind speed with the distribution of snails infected with F. hepatica or F. gigantica in the northwestern regions of Iran. In contrast to F. gigantica, F. hepatica was more prevalent in low-altitude areas. Further research is recommended to elucidate the relationship between geoclimatic factors and the presence of intermediate hosts of the two Fasciola species. Graphical Abstract


Background
Fascioliasis is a vector-borne disease transmitted by aquatic and amphibious snails and is caused by either Fasciola hepatica or Fasciola gigantica of the digenean Platyhelminthes [1].Fasciola hepatica is common in temperate zones such as Asia, Europe, the Americas, and Australia, while F. gigantica is limited to the tropical or subtropical regions of Africa, Asia, and the Far East.In contrast, both species overlap in occurrence in subtropical areas [2].
Fascioliasis is a common disease of livestock, and although it is rarer in humans, the estimated prevalence and projected disease burden of human fascioliasis are also significant [3].According to the World Health Organization (WHO), human fascioliasis has been reported in more than 70 countries, with at least 2.4 million people infected worldwide and several million more at risk (https:// www.who.int/ publi catio ns/i/ item/ WHO-UCN-NTD-VVE-2021.4).
Freshwater snails within the Lymnaeidae family are of great interest due to their participation in the life cycle of various trematodes with biomedical and veterinary importance [1,4].About 20 species of lymnaeid snails have been described as the potential intermediate hosts of Fasciola spp.Based on the research conducted so far, lymnaeid snails of the genera Galba and Lymnaea, especially Galba truncatula and Radix euphratica (syn.Radix gedrosiana), are known as the main first intermediate hosts of Fasciola spp. in different parts of the world [1].The epidemiology of fascioliasis is highly related to the ecological characteristics of their snail hosts, while the susceptibility of these intermediate hosts to Fasciola species may vary [5].Research has shown that F. hepatica is more common in high and mountainous areas, while F. gigantica is more common in lower areas, depending on the distribution of the intermediate snail [6,7].Controlling the population of intermediate snail hosts can be considered a suitable strategy to reduce the endemicity of food-borne trematode infections, especially fascioliasis [8][9][10].This issue shows the importance of investigating the influence of geographical factors on the distribution of intermediate snail hosts and their infection rate.This study was designed to investigate the influence of environmental and geoclimatic factors on the spread of lymnaeid snails infected with Fasciola in the northwestern regions of Iran, which constitute one of the most important centers of agriculture and animal husbandry in Iran, and where animal fascioliasis is also highly prevalent [11].

Study area
This study was conducted in the northwestern region of Iran, an area between latitudes 35.5511 and 39.7820 N and longitudes 44.0325 and 49.4337 E, which includes four provinces of West Azerbaijan, East Azerbaijan, Ardabil, and Zanjan (Fig. 1).
The study areas border Turkey and Iraq in the west; Armenia, the Republic of Azerbaijan, and the Nakhchivan Autonomous Republic of Azerbaijan in the north; Gilan Province, the most important center of human fascioliasis in Iran, in the east; and Kurdistan, Qazvin, and Hamedan Provinces in the south.The climate of the study area varies from Mediterranean and temperate climates in the plains to cold and snowy mountainous climate.

Snail collection and identification
Based on the epidemiological pattern of fascioliasis in Iran [11,12] and the distribution of lymnaeid snails in habitats [13,14], locations near the final hosts (livestock) such as rivers, swamps, bogs, riverbanks, ponds, creeks, streams, and marshes were selected for sampling.Freshwater lymnaeid snails were collected by searching 33 permanent and seasonal habitats in the northwestern provinces of Iran from June to November 2021.A total of 2000 lymnaeid snails were collected manually using a sweeping net (Table 1).The collected samples were placed in bottles containing snail habitat water and were transferred to the parasitology laboratory at the Medical School of Zanjan University of Medical Sciences, where they were identified morphologically according to the keys provided by Pfleger [15].Since it is not possible to differentiate some lymnaeid species such as G. truncatula and G. schirazensis by morphological methods [16], it was necessary to use the DNA sequencing method to differentiate these species.In this study, DNA sequencing of all snails was not possible, so the information about these two species is shown as G. truncatula/G.schirazensis.

Examination of cercariae from snails
After 2 days of acclimatization to laboratory conditions, the snails were divided into groups of eight inside Petri dishes containing 20 ml filtered water originating from a pond.These dishes were placed at 25-30 °C under artificial light for at least 2 h or overnight to induce shedding.After the incubation, the Petri dishes were examined under a stereomicroscope for the presence of cercariae.Snails from positive batches were tested individually to identify the infected snails.Emerged cercariae were collected in dechlorinated water and observed for their swimming behavior and then photographed and preserved in 96% ethanol at −20 °C for molecular investigations.Several cercariae isolated from each snail were stained with Ehrlich's alum hematoxylin and eosin and were classified using a light microscope according to Frandsen and Christensen [17].
Also, several collected snails were crushed against a glass plate and then examined with a stereomicroscope for trematode cercariae, rediae, or sporocysts.Snails infected with trematode larval stages were preserved in individual containers using 96% ethanol for molecular investigations.

DNA extraction and PCR-RFLP of ITS1 rDNA for Fasciola species identification
The ethanol-preserved cercariae were subjected to DNA extraction using a SamBio kit (Ambio ® , Sambio ™ , Iran), according to the manufacturer's instructions.Amplification of the internal transcribed spacer 1 (ITS1) fragment with genus-specific primers was used to identify Fasciola cercariae isolated from snails.PCR amplification of ITS1 of the ribosomal DNA (fragment 680 base pairs [bp] in length) was performed using primers ITS1-F (5′-TTG CGC TGA TTA CGT CCC TG-3′) and ITS1-R (5′-TTG GCT GCG CTC TTC ATC GAC-3′) [18].The PCR reaction was carried out in a total volume of 25 μl, containing 10 pmol of each primer, 5 μl template DNA, 10 µl of master mix (GeneDireX, Taiwan) and 8 μl double-distilled water (ddH 2 O).PCR conditions were adjusted by pre-denaturation at 95 °C for 4 min, denaturation at 95 °C for Fig. 1 Geographical location of the sampling areas for lymnaeid species in Iran.The provinces surveyed for this study are shown in dark green 60 s, annealing at 55 °C for 45 s, elongation at 72 °C for 60 s in 35 cycles, and post-elongation at 72 °C for 10 min.In each PCR run, ddH 2 O was used instead of the DNA template as negative control.The PCR products were separated on agarose gel (1.5%) and visualized using a transilluminator.Restriction fragment length polymorphism (RFLP) analysis was conducted to differentiate F. hepatica from F. gigantica in the ITS1 gene [19] using the RsaI restriction enzyme (Thermo, Germany).A total volume of RFLP reaction contained 1 μl RsaI enzyme, 1 μl buffer, 5 µl PCR product, and nuclease-free water for a final volume of 25 μl.The mixture was incubated at 37 °C for 16 h.The products were run on 2% agarose gel, stained with safe stain, and visualized by an ultraviolet (UV) transilluminator.PCR-RFLP produced the three expected restriction fragment sizes of 360, 100, and 60 bp for F. hepatica, and three fragment sizes of 360, 170, and 60 bp for F. gigantica [19] (Fig. 2).

Geospatial data
The latitude-longitude coordinates and altitude of the collection sites of Fasciola-infected and noninfected snails were retrieved from global positioning system (GPS) data (Premium GPS Map Camera version 1.3.6)and imported into an Excel file.The file was exported to ArcMap 10.5 (http:// www.esri.com/ arcgis) software and the attribute point shape file layer was generated.This layer encompassed all points where snails had been gathered.The weather temperature (mean, minimum, and maximum values), soil temperature, dew temperature, rainfall, humidity (mean, minimum, and maximum values), evaporation, air pressure (mean, minimum, and maximum values), and wind speed data were recovered from 57 weather synoptic stations for 1 year (2021) in the four provinces of Ardebil, Azarbayjan-e-Sharghi, Zanjan, and Azarbayjan-e-Gharbi.Digital elevation model (DEM) raster and land cover vector layers were recovered from the natural resources departments of the four investigated provinces.Then they were merged to generate one layer that covered the entire study area.

Geoclimatic analysis
After examination of errors of different interpolation methods, the raster layers of isothermal, isohydral, and evaporation were generated by the tension-based spline interpolation method.The different pressures and wind speed layers were made by kriging and humidity layer by inverse distance weighted (IDW) interpolation methods with a resolution grid of 2 × 2 km.The point shape file layer including 33 sampling points was extracted with the geoclimatic and DEM raster layers.The identity tool was used to calculate the geometric intersection of the layer obtained from the extraction of all raster layers with land cover (polygonal) vector layer to develop the final layer in which each point has the properties of all the overlapped identity features from the abovementioned raster and vector layers.The analysis was done using ArcGIS version 10.5.The attribute of this layer was converted to an Excel format and exported to SPSS software for statistical analysis.

Statistical analysis
Statistical analyses were performed between areas where snails infected with Fasciola cercariae were found and those with noninfected snails.They were also realized between areas with snails infected with F. hepatica and those with snails infected with F. gigantica using the t-test and Chi-square test.The analyses were performed using IBM SPSS version 21 software.

Results
The collected lymnaeid snails were identified as R. euphratica, G. truncatula/G.schirazensis, and L. stagnalis.PCR-RFLP of the ITS1 fragment of cercariae isolated from 38 lymnaeid snails showed that 19 snails were infected with F. hepatica and six snails were infected with F. gigantica (Table 1).
The three values of the meteorological temperature, soil temperature, and dew temperature between the points with uninfected snails and those with snails harboring Fasciola cercariae showed no significant differences.Similarly, average rainfall and the three values of moisture and evaporation between these geographical points were not different.The same applies to the three values of atmospheric pressure and wind speed.The average elevation between these points was not statistically different (Table 2).Finally, the Chi-square test showed no significant difference between points with uninfected snails and those with individuals harboring Fasciola cercariae compared to the land cover where these points are distributed (Table 3).
Statistical analyses of geoclimatic characteristics of F. hepatica-and F. gigantica-prevalent points showed that the mean humidity and maximum humidity were both different between those areas (P < 0.05), while minimum humidity, rainfall, and evaporation were not associated with the presence of Fasciola species.Compared with areas with F. gigantic-infected snails, the humidity was higher in F. hepatica-infected areas (Fig. 3).Moreover, wind speed showed a significant difference (P < 0.05) between F. hepatica-and F. gigantica-infected points, and it was higher in F. hepatica-infected areas.Although elevation was not significantly different between F. hepaticaand F. gigantica-infected points, a trend was found for this factor, as the areas of F. hepatica were about 300 m lower than those of F. gigantica (Fig. 4 and Table 4).Finally, no difference was found between F. hepatica-and F. gigantica-infected areas in terms of land cover (Fig. 4 and Table 5).

Discussion
Considering the role of snails in the transmission of important human parasites such as Fasciola, Schistosoma, and Clonorchis, it is very important to study the population distribution of host snails, the various factors affecting their distribution, and the extent of their contamination [20,21].The occurrence cycle of fascioliasis, as one of the most important diseases transmitted by snails, is largely related to snails of the Lymnaeidae family [1].Several studies have investigated the relationship between climate change and parasitic diseases, especially new and re-emerging diseases [22][23][24].According to the research, it seems that in fascioliasis, the geoclimatic and environmental characteristics have several effects on larval forms of Fasciola and on lymnaeid snails [25][26][27].Also, the relationship between the emergence of fascioliasis and aspects of global changes such as import/ export and livestock management, changes in the human environment, travel, and changes in human nutritional habits (diet) has been investigated [1,28,29].
Among the environmental and geoclimatic factors, altitude is an important factor in the prevalence of fascioliasis.Different results have been obtained in the studies on the effect of altitude on the spread of fascioliasis and Fasciola species.In a study using the geographical information system (GIS) in the south of Brazil, a higher percentage of infection with F. hepatica was reported in cattle in low-altitude areas, about 150 m above sea level [30], while the highest prevalence of human fascioliasis was reported in the very high-altitude areas of the northern Bolivian Altiplano at an altitude between 3800   and 4100 m above sea level [31].In the present study, fascioliasis was observed in lymnaeid snails at altitudes between 827 and 2319 m in the northwestern regions of Iran.Although no significant difference was observed between the elevation of F. hepatica-and F. giganticainfected areas, F. gigantica-infected areas were about 300 m higher than the F. hepatica-infected areas.However, in a study conducted on the relationship between altitude and the prevalence of F. hepatica and F. gigantica in the Nile River basin in Ethiopia, researchers using GIS modeling concluded that the infection rate of F. gigantica in livestock living in the area in question decreased with increasing elevation, while the opposite occurred in the case of F. hepatica [32].In a study by Ashrafi et al. [33] on fascioliasis in ruminants in Gilan province of Iran, the prevalence of F. gigantica in areas below sea level was significantly higher than that of F. hepatica, while in areas with an altitude of more than 100 m, the prevalence of F. hepatica was significantly higher than that of F. gigantica.Considering that in this study the intermediate host snails of these two species of Fasciola were not investigated, it is difficult to compare its results with the present research.However, it is possible that the difference in the environmental fauna and the average height of the study areas in these two studies influenced this difference.Also, the extreme events related to climate change in the study areas of the present research, which were responsible for the superiority of the distribution of R. euphratica as the main intermediate host of F. gigantica [34], should be considered.
Contrary to the results of the current study, the prevalence of F. gigantica in Ugandan buffaloes living below 1500 m was significantly higher than that in areas above 1500 m [35].These results could be related to the high prevalence of the parasite in Ligia natalensis, the intermediate host of F. gigantica, at low altitude in the area studied in Uganda.In comparison, it appears that the higher prevalence of F. gigantica in high-altitude Iranian areas is also related to the frequency of the host snail in this study.In fact, it can be argued that the prevalence of the two species of Fasciola in snails living at different altitudes is directly related to the ecology of the host snail.Temperature and humidity are also important influencing factors in the spread of fascioliasis.According to a report by Rojo-Vazquez et al. [36], the seasonality of fascioliasis is closely related to rainfall and temperature, and even slight changes in these factors may directly affect the life cycle of the parasite in the intermediate hosts.By modeling the effects of climate change and predicting the risk factors affecting the spread of F. hepatica using GIS, Fox et al. [37] concluded that fascioliasis outbreaks in areas with higher and longer annual precipitation, high soil moisture, and excess water will likely be more numerous.In the present study, humidity was found to be a determining factor in the spread of F. hepatica and F. gigantica, as humidity was significantly higher in areas infected with F. hepatica than in the areas where F. gigantica was found.In this research, the wind speed in the areas where the snails were infected with F. hepatica was significantly higher than the areas where the snails were infected with F. gigantica.Due to the lack of information about the effect of wind speed on the prevalence of fascioliasis, there is a need for more research on this topic in fascioliasis-endemic areas.The analysis of other environmental and climatic factors investigated in the present study did not show any significant difference between the areas infected with fascioliasis and those not infected with it, and between the two species of Fasciola studied.
According to Bargues et al. [16], the high phenotypic similarity and genotypic differences between G. schirazensis and G. truncatula is an important issue in the molecular epidemiology of fascioliasis.These researchers consider it necessary that a large body of literature on G. truncatula should be revised; although G. truncatula is a known host of F. hepatica, the role of G. schirazensis as a host for Fasciola spp.has not been proven [20].Therefore, the sympatric distribution of these two species of snails can interfere in estimating the epidemiology of fascioliasis [38].Although in this study the molecular differentiation of these two species of lymnaeid snails was not performed, it seems not to affect the final analysis of bioclimatic factors on the prevalence of fascioliasis in these snails.

Conclusions
Most of the research conducted on the relationship between climatic and geographical factors with the prevalence of different Fasciola species has been done on the final hosts of the parasite.However, considering the direct relationship of many of these factors with the ecology and biological conditions of the snail host, it seems that more research is needed in this field.According to this aim, the present study shows the relationship between a number of climatic and geographical factors including humidity, wind speed, and to some extent altitude with the differences in the prevalence of F. hepatica

Fig. 4
Fig. 4 Maps of the environmental factors: digital elevation model (a) and land cover (b)

Table 2
Results of geoclimatic factors of Fasciola-infected and noninfected points

Table 3
Distribution of uninfected and Fasciola-infected points in different types of land coverRangeland: Grasslands, shrublands, woodlands, wetlands, and deserts that are grazed by domestic livestock or wild animals

Table 4
Geoclimatic characteristics of farms where snails infected with F. hepatica and F. gigantica were found SD standard deviation.*Indicates significance at P < 0.05

Table 5
Frequency of grazing types on farms infected with F. hepatica and F. gigantica F. gigantica in the northwestern regions of Iran.Further research is recommended on the relationship between environmental and climatic factors and the contamination of intermediate host snails with different Fasciola species. and